CN113980966A

CN113980966A - Nucleic acid sequence for inhibiting PCSK9 target gene expression and application thereof

Info

Publication number: CN113980966A
Application number: CN202111168503.6A
Authority: CN
Inventors: 张必良; 李曼
Original assignee: Agna Biopharmaceutical Co ltd
Current assignee: Agna Biopharmaceutical Co ltd
Priority date: 2021-09-29
Filing date: 2021-09-29
Publication date: 2022-01-28

Abstract

The invention relates to siRNA for inhibiting PCSK9 gene expression, wherein the sense strand nucleotide sequence is shown as SEQ ID NO.1, the antisense strand nucleotide sequence is shown as SEQ ID NO.3, and the siRNA generated by annealing a new nucleotide sequence formed by respectively substituting, deleting and/or adding 1-6 bases in the sense strand nucleotide sequence and the antisense strand nucleotide sequence is structurally modified by various chemical modification methods, so that the siRNA has good inhibition activity on the expression of the gene PCSK 9. The siRNA is subjected to ligand modification to form a new conjugate, and the conjugate has high inhibition activity and stability, has high liver targeting property and capacity of promoting endocytosis of cells, has excellent affinity and provides possibility for future targeted therapy.

Description

Nucleic acid sequence for inhibiting PCSK9 target gene expression and application thereof

Technical Field

The invention relates to the field of molecular biology, in particular to a nucleic acid sequence for inhibiting PCSK9 target gene expression and application thereof.

Background

The Proprotein convertase subtilisin/kexin type 9 (PCSK 9) belongs to Ca²⁺Dependent on serine endoprotease, PCSK9 can pass through extracellularly andthe intracellular pathway is combined with a Low Density Lipoprotein Receptor (LDLR) to promote the degradation of the LDLR; the extracellular pathway is also the main way to function, PCSK9 secreted to the outside of the cell is degraded by binding with LDLR on the cell membrane, causing LDLR to be endocytosed to form an endosome, eventually entering lysosome; PCSK9 secreted from the golgi apparatus in the intracellular pathway enters the cytoplasm and directly binds to LDLR, which leads LDLR to directly enter the lysosomal degradation pathway in the cell. Since LDLR is an important way for tissues, especially the liver, to take up circulating cholesterol, degradation of LDLR by increased levels of PCSK9 results in increased levels of circulating cholesterol, making PCSK9 an important target for lowering blood cholesterol.

In addition, the rise of low-density lipoprotein cholesterol (LDL-C) is a main risk factor of cardiovascular diseases, and the PCSK9 inhibitor has a strong LDL-C reducing effect and is a novel promising lipid-lowering medicament. The siRNA medicament targeting PCSK9 blocks the function of PCSK9 protein from mRNA level, reduces LDL-C level in blood, and has the characteristics of good specificity, easy design, low toxicity, no drug resistance and the like compared with the traditional medicament.

siRNA has great development potential as a new treatment method, siRNA acts on mRNA in cells, and compared with the traditional small molecule drugs, siRNA can directly silence target genes, so that occurrence and development of tissue diseases can be fundamentally and more efficiently realized. The siRNA is properly modified to increase the stability of the siRNA and effectively inhibit the expression of a target gene.

Disclosure of Invention

Based on the above, one of the objects of the present invention is to provide an siRNA which inhibits or reduces PCSK9 target gene expression.

The technical scheme is as follows:

an siRNA that inhibits or reduces the expression of a PCSK9 target gene, consisting of a complementary sense strand and antisense strand; the base composition sequence of the sense strand is selected from SEQ ID NO.1-SEQ ID NO.2, and the 1 st base from the 5 ' end and the 1 st-2 nd continuous base from the 3 ' end of the sense strand are both subjected to 2' -O-methyl modification; the base composition sequence of the antisense chain is selected from SEQ ID NO.3-SEQ ID NO. 9.

One of the objects of the present invention is also to provide a pharmaceutical composition.

The technical scheme is as follows:

the effective component of the pharmaceutical composition comprises the siRNA for inhibiting or reducing the expression of the PCSK9 target gene.

The invention also aims to provide application of the pharmaceutical composition in preventing or alleviating or treating diseases caused by expression of the PCSK9 target gene.

It is also an object of the present invention to provide a method for inhibiting or reducing PCSK9 target gene expression.

The technical scheme is as follows:

a method for inhibiting or reducing the expression of a PCSK9 target gene is to introduce the siRNA for inhibiting or reducing the expression of the PCSK9 target gene and/or the pharmaceutical composition.

Based on the intensive research on PCSK9 and siRNA technology, the inventor finds an siRNA with a sense strand base sequence of SEQ ID NO.1 and an antisense strand base sequence of SEQ ID NO.3 or with a sense strand nucleotide sequence and an antisense strand nucleotide sequence of SEQ ID NO.8 through replacement, deletion and/or addition of a plurality of bases and modification, and can reduce the degradation of the siRNA by nuclease in blood and tissues on the premise of keeping the capability of inhibiting the expression of a target gene, increase the stability in vivo and improve the applicability of the siRNA as a clinical drug. In particular, the above-mentioned siRNA can be obtained by modifying a part of the bases in the above-mentioned base sequence in a specific chemical combination (i.e., the sense strand is modified from the 5 'end to the 3' end, the 1 st, 2 nd, 6 th bases are modified with 2 '-O-methyl, the 4 th, 8 th, 10 th bases are modified with 2' -fluoro, the penultimate 1 st, 2 nd, 5 th, 6 th, 8 th bases are modified with 2 '-O-methyl, the penultimate 3 rd, 4 th, 7 th bases are modified with 2' -fluoro, and the antisense strand is modified with only 2 '-fluoro, in particular, the 1 st, 4 th, 5 th, 7 th, 8 th, 10 th, 12 th, 13 th, 14 th, 16 th, 17 th, 20 th, 21 th, 22 th, 24 th bases are modified with 2' -fluoro) in the direction from the 5 'end to the 3' end): RB-51 was able to effectively reduce PCSK9 target gene expression at concentrations as low as 6 nM.

For siRNA: RB-51 was further ligand modified, in the formed conjugate (GalNac-siRNA): in RB59-RB71, an endocytosis experiment and an affinity experiment show that RB-61 and RB-70 can effectively reduce the expression of a PCSK9 target gene, have good capacity of promoting endocytosis of cells, and have excellent affinity, so that the influence on other tissues or organs can be reduced and the use amount of siRNA molecules can be reduced in clinical use, and the aims of reducing toxicity and reducing cost are fulfilled; can also enter cells or tissues more effectively to play a role, is expected to be developed into lipid-lowering medicaments and provides possibility for targeted therapy.

Drawings

FIG. 1 is a statistical result of the effect of different siRNAs on PCSK9mRNA expression level in example 1.

FIG. 2 is a reaction scheme for the synthesis of galactose-modified monomer L in example 2.

FIG. 3 is a statistical result of the effect of different siRNAs on PCSK9mRNA expression level in the experiment of example 3 in which HeLa cells were used for endocytosis.

Figure 4 is a statistical result of the effect of different conjugates on PCSK9mRNA expression levels using primary mouse hepatocytes for endocytosis experiments in example 3.

FIG. 5 is the MFI statistics of the mean fluorescence intensity of Cy5 fluorescence of live cell populations under different conjugates for endocytosis experiments using primary mouse hepatocytes as in example 3.

Detailed Description

In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It is to be understood that the experimental procedures in the following examples, where specific conditions are not noted, are generally in accordance with conventional conditions, or with conditions recommended by the manufacturer. The various reagents used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The present invention will be described in further detail with reference to specific examples.

Some embodiments of the invention provide an siRNA that inhibits or reduces PCSK9 target gene expression, consisting of a complementary sense strand and antisense strand; the base composition sequence of the sense strand is selected from SEQ ID NO.1-SEQ ID NO.2, and the 1 st base from the 5 ' end and the 1 st-2 nd continuous base from the 3 ' end of the sense strand are both subjected to 2' -O-methyl modification; the base composition sequence of the antisense chain is selected from SEQ ID NO.3-SEQ ID NO. 9.

In some embodiments, the siRNA for inhibiting or reducing PCSK9 target gene expression can make the siRNA more resistant to nuclease hydrolysis after introducing a substituent to modify hydroxyl at the 2' position of ribose. And the discovery shows that the siRNA modified by 2' -O-methyl can effectively avoid the complete methylation of any chain in the siRNA, thereby avoiding the loss of the gene silencing activity of the siRNA; the siRNA modified by 2' -fluorine makes the siRNA difficult to be identified by RNase, thereby increasing the stability of the siRNA.

In some embodiments, in the siRNA for inhibiting or reducing expression of a PCSK9 target gene, the sense strand has 2 '-O-methyl modifications at bases 1 to 7 and 2' -O-methyl modifications at bases 1 to 7 from the last in the direction from the 5 'end to the 3' end; or, the 1 st and 6 th bases are modified with 2' -O-methyl, the 1 st, 2 th, 5 th and 6 th bases are modified with 2' -O-methyl, and the 4 th and 8 th bases are modified with 2' -fluoro.

In some embodiments, the siRNA for inhibiting or reducing expression of a PCSK9 target gene comprises an antisense strand having a 2' -O-methyl modification at base 2 in the 5 ' to 3 ' direction; or the 3 rd, 4 th, 7 th and 8 th bases are deoxyribonucleotide modifications; or the 9 th, 11 th, 13 th, 14 th, 15 th, 17 th and 18 th bases are 2' -fluoro modified.

In some embodiments, the siRNA formed by the sense and antisense strands described above comprises RB1-RB 57.

In some embodiments, the siRNA for inhibiting or reducing PCSK9 target gene expression has a sense strand with the base composition sequence of SEQ ID No.1, and the sense strand has 2 '-O-methyl modification at 1, 2, 6 th base, 2' -fluoro modification at 4, 8, 10 th base, 2 '-O-methyl modification at the 1, 2, 5, 6, 8 th base from the last base, and 2' -fluoro modification at the 3, 4, 7 th base from the 5 'end to the 3' end; the base composition sequence of the antisense chain is SEQ ID NO.8, and the 1 st, 4 th, 5 th, 7 th, 8 th, 10 th, 12 th, 13 th, 14 th, 16 th, 17 th, 20 th, 21 th, 22 th and 24 th bases of the antisense chain are 2' -fluoro-modified from the 5 ' end to the 3 ' end.

In some embodiments, the siRNA for inhibiting or reducing expression of the PCSK9 target gene has a sense strand of mcmadgfugdfugdud gmafcmamgmu and an antisense strand of afugafcfugufafcafcaffcufugfcufugfcufugufu. Wherein, N: the generic term unmodified ribonucleotide includes the compounds represented by A: adenine ribonucleotide, G: guanine ribonucleotide, C: cytosine ribonucleotides, U: uracil ribonucleotides. dN: the generic term unmodified deoxyribonucleotides includes dA: adenine deoxyribonucleotide, dG: guanine deoxyribonucleotide, dC: cytosine deoxyribonucleotide, dT: thymine deoxyribonucleotides. fN: 2' -fluoro-modified ribonucleotides. mN: 2' -O-methyl modified ribonucleotides.

In some embodiments, the siRNA that inhibits or reduces PCSK9 target gene expression has a ligand modification that improves cellular uptake, intracellular targeting, half-life, or pharmacokinetic or kinetic properties of the siRNA molecule. In some embodiments, the ligand-modified siRNA has enhanced affinity or cellular uptake for a selected target (e.g., a particular tissue type, cell type, organelle, etc.), preferably hepatocytes, as compared to an siRNA that is not modified with a ligand, and does not interfere with the activity of the siRNA.

In some embodiments, the ligand modification is at the sense strand end or the 3' end of the antisense strand of the siRNA.

In some embodiments, the siRNA that inhibits or reduces PCSK9 target gene expression described above may be modified with a ligand to form a conjugate.

In some embodiments, the ligand is modified by connecting the siRNA for inhibiting or reducing the expression of the PCSK9 target gene with a ligand compound, wherein the ligand compound has the structure of Ax-linker-R.

In some of the above ligand-modified ligand compound structures, the Ax is selected from the structures in the following table:

in some embodiments, the linker is selected from any of the following structures:

wherein n is an integer of 1 to 10, more preferably, n is 1;

wherein n is₁And n₂Each independently selected from integers of 1 to 10, more preferably n1 is 1 and n2 is 1;

wherein n is₁、n₂、n₃Each independently selected from integers of 1 to 10, more preferably, n1 is 1, n2 is 1, n3 is 1;

wherein n is an integer of 1 to 10, and more preferably, n is 1.

In some embodiments, in the above ligand-modified ligand compound structures, the R is selected from any one of the following structures:

R₁is composed of

R₂Is composed of

R₂M in₁And m₂Each independently selected from an integer of 1 to 10,

R₃is composed of

R₁、R₂、R₃Wherein Z is a protecting group for a hydroxyl group, preferably, each Z is independently 4, 4-dimethoxytrityl or 4-methoxytriphenylchloromethylmethyl.

In some embodiments, the ligand compound has the structure:

wherein n is an integer from 1 to 10.

In some embodiments, n in the structure of the ligand compound is 1, and the ligand compound is galactose modified monomer L, referred to as complex L.

In some embodiments, the ligand compound is modified by the coordination with siRNA to have any of the following structures:

wherein Nu is the nucleotide sequence in the siRNA for inhibiting or reducing the expression of the PCSK9 target gene.

In some embodiments, the conjugate formed by the above-mentioned coordination modification, the nucleotide sequence can be selected from the sense strand of the siRNA shown in SEQ ID NO.1-SEQ ID NO.2 and/or the antisense strand sequence shown in SEQ ID NO.3-SEQ ID NO.9, or the modified sense strand and antisense strand sequences of the siRNA in RB1-RB 57.

In some embodiments, the conjugate formed by the above conjugate modification is a sense strand mcmaddgfafaaddgudgafgdudgmafcmamugmu of siRNA or an antisense strand afugafufafcafcafcaffcufugfcfcfcfcfcfcfcfcfcfcfcfcfcufugfcu.

In some embodiments, the 3 'end or the 5' end of the siRNA can form a conjugate with a ligand compound, the structure of the conjugate formed by the siRNA and L can be referred to as ZW1001, the structure of the conjugate formed by the siRNA and LL can be referred to as ZW1002, and the structure of the conjugate formed by the siRNA and LLL can be referred to as ZW 1003.

In some embodiments, the above ligand compound is modified by the coordination of siRNA to ZW1003 or ZW1004, wherein n in the ligand compound is 1.

In some embodiments, the conjugate formed by the complex modification further comprises a fluorescent label and/or a biotin label; the fluorescently labeled fluorophore includes, but is not limited to, FAM, TET, JOE, HEX, Cy3, TAMRA, ROX, Texas, Red, LC Red640, Cy5, LC Red705, Alexa Fluor488, and Alexa Fluor 750, preferably Cy 5; the biotin label can reduce steric hindrance effect and increase the sensitivity and specificity of detection. The biotin used includes, but is not limited to, N-hydroxysuccinimide ester (BNHS), p-nitrophenol ester (pBNP), Biotin Hydrazide (BHZ) and hydrazinized Biotin (BCHZ).

In some of these embodiments, the conjugate formed by the above-described complexing modifications is shown as RB58-RB 71.

In some of these embodiments, the conjugate is RB-61, with the sense strand being mCMADGfCdAdGfUdGfGdGmAfCmAmGfCmAmGfCmAmU-LLL and the antisense strand being/cy 5/-AfUGAfCfUGfUfCAfCfUGfCfUGfGfCfCfCfUGfUGfU; or RB-66, wherein the sense strand is LLL-mCMADGfGfCdAdGfUdGmAfCmAmGfUfCmAm mAm mU, and the antisense strand is/cy 5/-AfUGAfCfUGfUfCAfCfUfUGfCfCfUGfU; or RB-66, wherein the sense strand is mCMADGfCdAMadAdGfGfUdGmAfCmAmGfUfCmAmU-LLLL, and the antisense strand is/cy 5/-AfUGAfCfUGfUfCAfCfUfUGfUGfU. Wherein,/cy 5/is cy5 fluorescent dye label; l is the L-type ligand compound.

Some embodiments of the present invention provide a pharmaceutical composition, the effective ingredient of which comprises the above-mentioned siRNA for inhibiting or reducing PCSK9 target gene expression.

In some embodiments, the pharmaceutical composition can be applied to diseases mediated by the PCSK9 gene, including but not limited to hyperlipidemia, hypercholesterolemia, melanoma and liver cancer.

Some embodiments of the invention also provide a method of inhibiting or reducing expression of a PCSK9 target gene in a cell, comprising introducing into the cell an siRNA and/or pharmaceutical composition of the invention. As used herein, the term "introduced" refers to facilitating uptake or absorption into a cell, which may occur through non-ancillary diffusion or active cellular processes, or through ancillary reagents or devices. In addition to the use of transfection reagents, other known methods for delivering siRNA molecules into cells can be used, such as injection, transfection with vectors (which can be plasmids or viruses), electroporation, lipofection, and the like.

In some embodiments, the method comprises inhibiting or reducing PCSK9 target gene expression by:

(1) obtaining a cell to be transfected and the siRNA for inhibiting or reducing the expression of the PCSK9 target gene;

(2) mixing the siRNA with a cell to be transfected and transfecting the cell;

(3) and (5) culturing the cells to obtain the compound.

In some of these embodiments, the concentration of siRNA transfection in the above methods is 4nM to 0.1. mu.M; more preferably 6nM to 60 nM.

In some embodiments thereof, the cells in the above methods are mammalian cells expressing PCSK9, preferably high levels of expression of the PCSK9 gene in target cells, e.g., primate cells, such as human cells; preferably, the cell is derived from brain, salivary gland, heart, spleen, lung, liver, kidney, intestinal tract or tumor. More preferably a liver cancer cell or a cervical cancer cell, and still more preferably a HeLa cell.

Example 1 preparation and detection of PCSK9-siRNA

First, siRNA design

Based on the human PCSK9mRNA sequence, multiple pairs of PCSK 9-siRNAs are designed by selecting different sites, all the designed single siRNAs can target all transcripts of target genes (as shown in Table 1), and the multiple pairs of siRNAs have the lowest homology with all other non-target gene sequences through sequence similarity software alignment. Sequence design methods are referenced to Elbashir et al.2002; paddison et al.2002; reynoldset al.2004; the method of Ui-Tei et al 2004 et al.

TABLE 1-1

Target genes	Species (II)	GeneID	NM_ID
				PCSK9	Homosapiens (human)	255738	NM_174936.3

Second, siRNA Synthesis

The basic base sequence (unmodified) of the siRNA related to the invention is shown in the following tables 1-2:

tables 1 to 2

The siRNA sequences formed by the combination of the sense strand and the antisense strand in the table after being independently modified are shown in the following tables 1 to 3, and the specific preparation steps refer to Chinese patent CN 109957567A:

TABLE 1-3 siRNA sequence Listing

Wherein, the abbreviations in the sequence have the following meanings:

n: the generic term unmodified ribonucleotide includes the compounds represented by A: adenine ribonucleotide, G: guanine ribonucleotide, C: cytosine ribonucleotides, U: uracil ribonucleotides.

dN: the generic term unmodified deoxyribonucleotides includes dA: adenine deoxyribonucleotide, dG: guanine deoxyribonucleotide, dC: cytosine deoxyribonucleotide, dT: thymine deoxyribonucleotides.

fN: 2' -fluoro-modified ribonucleotides.

mN: 2' -O-methyl modified ribonucleotides.

Third, siRNA inhibits PCSK9 gene expression level

Experimental materials: hela cells (

CRM-CCL-2^TM)，RiboFECT^TMCP Transfection Kit (Ribobio, C10511-1), NC1(Ribobio), NC2(Ribobio), antiserum-reduced Medium Opti-MEM (Gibco, 31985-^TMReverse Transcription Kit (Ribobio, C11027-2), 24-well plate (Corning, 3524), CO₂Incubators (Memmert, INC246), fluorescent quantitative PCR instruments (Bio-Rad, CFX96), and the like.

The experimental steps are as follows:

1. cell plating

The 24-well plate was rinsed with 1ml of PBS, and Hela in a good cell state was counted by normal digestion, and after diluting with DMEM complete medium, 50,000 cells per well were added, in a volume of 700uL per well. 37 ℃ and 5% CO₂The incubator was incubated overnight.

2. Cell transfection

Cell density was determined prior to transfection (approximately 50% or so, relatively uniform). All siRNA samples were configured into 20uM stock solutions before transfection, except for the test group, a normal cell control group (unotreated), a transfection reagent control group (Mock), a negative control group 1(NC1, irrelevant siRNA), a negative control group 2(NC2, irrelevant siRNA) were set. For each siRNA, 3 duplicate transfection systems were prepared as follows:

(1) a20 uM siRNA sample of 12uL was added to 138uL of Opti-MEM and mixed well.

(2) RiboFECT taking 15uL^TMCP transformation Reagent added to 135uL RiboFECT^TMMixing with CP Buffer.

(3) And mixing the diluted siRNA and the diluted transfection reagent, gently mixing uniformly, performing instantaneous centrifugation, and standing for 5-15min to prepare a transfection compound.

(4) The mixed transfection system was added to 3 replicate wells at 100 uL/well and mixed well with a final siRNA concentration of 100 nM.

(5) Cells were plated at 37 ℃ in 5% CO₂The incubator is used for 48 hours.

3. Extraction of RNA

Total RNA was extracted according to MagZol Reagent (Magen, R4801) instructions.

4. Fluorescent quantitative PCR and data analysis

(1) Reverse transcription PCR

Respectively adding the following components into a PCR tube to prepare a reverse transcription reaction system:

after the transient centrifugation, the reaction was carried out in a PCR instrument.

(2) The reaction was carried out at 42 ℃ for 60 min.

(3) The reverse transcriptase was inactivated at 72 ℃ for 10 min.

(4) Diluting by 5 times, mixing, and storing in refrigerator at-20 deg.C.

(2) Fluorescent quantitative PCR

Human housekeeping gene actin is used as an internal reference gene, and the sequence of an actin gene upstream primer is as follows: 5'-TCAAGATCATTGCTCCTCCTGAG-3' (SEQ ID NO.10), downstream primer sequence: 5'-ACATCTGCTGGAAGGTGGACA-3' (SEQ ID NO. 11); human PCSK9 gene upstream primer sequence: 5'-AAGCCAAGCCTCTTCTTACTTCA-3' (SEQ ID NO.12), downstream primer sequence: 5'-CCTGGGTGATAACGGAAAAAG-3' (SEQ ID NO. 13). The method comprises the following steps of performing real-time fluorescent quantitative PCR reaction by using 2X SYBR Green Mix:

the following components were added to a 96-well plate to prepare a PCR system, and each sample was repeated 3 times:

the PCR reaction was performed using a CFX96 fluorescent quantitative PCR instrument from Bio-Rad, USA, and the PCR reaction program was as follows:

after the PCR reaction, the temperature is increased from 70 ℃ to 95 ℃ to draw a melting curve so as to judge the correctness of the amplified product.

(3) Data analysis

After the PCR reaction is completed, the Ct error for 9 replicates of a sample (3 replicates for each individual sample at transfection and 3 replicates for each replicate at qpCR) should be. + -. 0.5. Relative quantitative analysis was then performed using CFX 2.1 software. The results of the analysis are shown in FIG. 1 and tables 1-4 below:

tables 1-4 mRNA expression levels of siRNA

Through the above statistical analysis of the real-time quantitative PCR detection results, in Hela cells, the siRNA molecules: RB1-RB57 can effectively inhibit the expression of the PCSK9 gene in cells, wherein the expression level of the PCSK9 gene in the cells transfected with RB1-RB6, RB8, RB10-RB14, RB17-RB22, RB24-RB27, RB47-RB48 and RB51 is lower than 0.1, which indicates that the corresponding siRNA has higher activity of inhibiting the expression of the PCSK9 gene.

Wherein, among RB1-RB3, RB5-RB7 and RB10-RB12, RB1, RB5 and RB10 inhibit PCSK9 activity to the highest degree, which indicates that the length of the antisense chain has an influence on the anti-PCSK 9 activity, and under the condition that the two ends of the sense chain are 7 methoxyl modifications or the 16 th nucleotide is added with methoxyl modification, the activity of the antisense chain with the length of 25 nucleotides is higher than that of siRNA with the length of 22 and 19 nucleic acids; RB19 activity in RB19-RB21 and RB23-RB25 is not as good as that in RB20 and RB 3538 and 21, and RB25 activity is better than that in RB23 and RB24, which shows that the activity of the antisense chain with the length of 19 nucleotides is higher than that of siRNA with the length of 25 nucleotides under the condition that two ends of the sense chain are modified by 7 methoxy groups or fluorinated modifications are added at the 10 th and 14 th nucleotides, and the 12 th position is modified by methoxy groups; RB4, a similar or slightly reduced anti-PCSK 9 activity relative to RB1, RB13 relative to RB10, RB22 relative to RB19, indicating that methoxy modification at nucleotide 2 of the antisense strand has little effect on its activity; RB5, RB-6 relative to RB1, RB14 relative to RB10, RB23 relative to RB19, are comparable in anti-PCSK 9 activity, indicating that the first 8 nucleotide modifications performed maintain their inhibition of PCSK9 activity; RB-8 activity is better than RB9, RB17 is better than RB18, which shows that under the condition that two ends of a sense strand are 7 methoxy modifications, the ribose ring of the 1 st, 3 rd and 4 th nucleotides of an antisense strand is added with a deoxy modification, the ribose ring of the 5 th nucleotide is replaced with a fluorinated modification, the 6 th and 8 th nucleotides are replaced with dT instead of fU to reduce the activity of siRNA against PCSK9, RB27 activity is better than RB26, which shows that two ends of the sense strand are 7 methoxy modifications, the 10 th and 14 th nucleotides are added with a fluorinated modification, under the condition that the 12 th nucleotide is added with a methoxy modification, the ribose ring of the 1 st, 3 th and 4 th nucleotides of the antisense strand is added with a deoxy modification, the ribose ring of the 5 th nucleotide is replaced with a fluorinated modification, and the 6 th and 8 th nucleotides are replaced with dT instead of fU to improve the activity against PCSK 9; RB47 activity was superior to RB48, indicating that in the case where the sense strand was modified at both ends with 8 methoxy groups, the ribose ring at nucleotides 9, 11, 13, 15, and 17 was modified with deoxygenization, the positions 10 and 14 were modified with fluorination, and the positions 12 and 16 were modified with methyloxidation, siRNA activity of 25 nucleotides in length of the antisense strand was superior to 19 nucleotides, and RB-51 activity was superior, indicating that the sense strand was modified at 19 nucleotides in length, both ends were modified with 2 methoxy groups, the positions 3, 5, 7, 9, and 11 were modified with deoxygenization, the positions 4, 8, 10, 13, 16, and 17 were modified with fluorination, and the positions 6, 12, 14, and 15 were modified with methylation, and the antisense strand was modified at 25 nucleotides in length, and the positions 2, 5, 6, 8, 9, 11, 13, 14, 15, 17, 18, 21, 22, 23, and 25 were modified with fluorination, anti-PCSK 9 activity was superior.

EXAMPLE 2 preparation of conjugates

1. Preparation of ligand compound: the synthetic reaction scheme of the galactose modified monomer L is shown in FIG. 2, and the specific preparation steps refer to the Chinese patent CN 109957567A.

2. Ligand compound modified siRNA

RB51 is modified by the complex L to form a conjugate, and the conjugate is subjected to biotin and fluorescence labeling on the part of the conjugate with reference to Chinese invention patent CN109957567A to form RB-58-RB-71, wherein the specific structure is shown in Table 2-1. Taking RB-59 as an example, the preparation steps are as follows:

TABLE 2-1

Wherein,/bio/: labeling with biotin; /cy 5/: cy5 fluorescent dye label; l: an L-form ligand compound.

Example 3 endocytosis assay

First, Hela cell

Experimental materials: hela cells, RiboFECT^TM CP Transfection Kit(Ribobio，C10511-1)，NC1(Ribobio，siB0943083707)，NC2(Ribobio，siB0818164933)，Biotin labeled mimicNC (Ribobio), antiserum-reduced Medium Opti-MEM (Gibco, 31985-^TMReverse Transcription Kit (Ribobio, C11027-2), 24-well plate (Corning, 3524), CO₂Incubators (Memmert, INC246), fluorescent quantitative PCR instruments (Bio-Rad, CFX96), and the like.

The experimental steps are as follows:

1. cell plating: corresponding procedure as in reference example 1

2. Cell transfection

The cell density was determined to be about 40-50% prior to transfection and was relatively uniform. All siRNA samples were configured as 20uM stock solutions before transfection, except for the experimental groups (e.g., RB-51 without ligand modification, RB-62 with galactose-modifying monomer L modification, RB-59 with galactose-modifying monomer L modification, and Biotin modification), and a normal cell control group (unotreated), a transfection reagent control group (Mock), a negative control group 1(NC1), a negative control group 2(NC2), and an endocytosis negative control group (Biotin labeled mic NC, NT) were set. The concentration of the samples after transfection was 100nM and the concentration of the untransfected samples was 500 nM. For each siRNA, 3 duplicate transfection systems were prepared according to the corresponding procedure in example 1:

3. cell incubation

The cell density is about 40-50%, and the cell density is relatively uniform. For each siRNA (3 replicate wells) dilutions were performed as follows:

(1) 60uL of 20uM siRNA samples were added to 240uL Opti-MEM and mixed well.

(2) The diluted siRNA was added to 3 replicate wells at 100 uL/well, a final concentration of 500nM and mixed well. Cells were plated at 37 ℃ in 5% CO₂The incubator is used for 48 hours.

4. Extraction of RNA

5. Fluorescence quantitative PCR and data analysis:

referring to the corresponding procedure in example 1, the results are shown in FIG. 3 and the following Table 2-1:

TABLE 3-1 mRNA expression levels of conjugates

According to the information in the table 2-1 and fig. 3, it can be known that RB-51 without ligand modification, RB-62 with galactose modification monomer L modification, RB-59 with galactose modification monomer L and biotin modification can significantly and effectively inhibit PCSK9 gene expression in cells under the condition of cell transfection, and compared with an endocytosis negative control group, the GalNAc-siRNA designed by the invention has the activity of inhibiting PCSK9 gene expression in cells under the condition of no cell transfection, so that the GalNAc-siRNA can sufficiently reduce PCSK9 target gene expression.

II, primary mouse liver cell endocytosis GalNac-siRNA

Experimental materials: mouse primary hepatocytes, Propidium iodide (sigma, P4170), TDL5M bench-top low-speed frozen centrifuge, U-bottom 96-well plate, flow cytometer (BDAccuri C6), multichannel pipettor, conjugate (GalNac-siRNA): RB-60, RB-61, RB-64, RB-65, RB-66, RB-67, RB-68, RB-69, RB-70, RB-71, all synthesized by Ruibo Biotech, Inc., Guangzhou.

The experimental steps are as follows:

1. cell preparation and identification

Anaesthetizing male C57BL/6 mouse, fixing on operation table, removing blood after puncture of hepatic portal vein, perfusing digestive fluid to liver, separating intact liver tissue after full digestion, placing in sterile culture dish containing stop solution, preparing into hepatocyte suspension, adding Percoll separating fluid at 4 deg.C, centrifuging at 50g for 2min to remove dead cells, washing cells for 2 times with DMEM culture medium containing 2% FBS, and diluting with DMEM culture medium containing 2% FBS to obtain diluted cell concentration of 4 x 10⁵one/mL. 1:1 dilution with tepralene blue dye and suspension of liver cells, cell count under electron microscope, cell viability (viable cell count/total cell count 100%)>At 70%, the subsequent test operations were carried out.

2. Mixed loading and incubation

Each GalNac-siRNA was plated in 3 duplicate wells, 24. mu.L of GalNac-siRNA (1. mu.M) was diluted in 176. mu.L of DMEM medium containing 2% FBS, mixed well and mixed with 200. mu.L of cells, and added to 3 wells of a U-bottom 96-well plate at a final siRNA concentration of 60nM for 100. mu.L per well.

mu.L GalNac-siRNA (0.1. mu.M) was diluted in 176. mu.L DMEM medium containing 2% FBS, mixed well and mixed with 200. mu.L cells, and added to 3 wells of a U-bottom 96-well plate at a final siRNA concentration of 6nM for 100. mu.L per well.

The U-shaped bottom 96-well plate was placed on ice, placed on a shaker, and incubated for 2h in the dark.

3. Detection of

The U-bottom 96-well plate was centrifuged at 50g for 2min at 4 ℃. 80uL of the supernatant was aspirated with a gun, 10ug/mL of Propidium iodide was prepared in PBS containing 2% FBS, and 100uL of PI reagent was added to each well and stained for 15 min. After staining was complete, the U-bottom 96-well plate was centrifuged at 50g for 2min at 4 ℃. After 2 times of washing with PBS containing 2% FBS, 100uL of PBS containing 2% FBS was added for resuspension, followed by flow cytometry, and the mean fluorescence intensity of Cy5 fluorescence of the viable cell population was counted, and the specific statistical results are shown in fig. 4 and the following table 3-2:

TABLE 3-2 mean fluorescence intensity MFI

From the results, it was found that RB-64 and RB-68 did not have endocytosis activity with the negative control RB-60 in which the ligand compound was not linked, and that the siRNA having high endocytosis activity were RB-67, RB-70, RB-61 and RB-66. The siRNAs with lower endocytic activity were RB-65, RB-69 and RB-71. Thus, it is shown that different numbers of galactose modified monomers L form specific conjugate (GalNac-siRNA) structures through being combined with siRNA, the endocytosis efficiency of the conjugate is obviously different, the endocytosis efficiency of the conjugate is higher when the number of the non-linked L monomers is larger, and the endocytosis activity of the conjugate is higher when only the number of the linked L monomers is 3 or 4.

Example 4 affinity assay

Experimental materials: mouse primary hepatocytes, Propidium iodide (sigma, P4170), TDL5M bench-top low-speed frozen centrifuge, U-bottom 96-well plate, flow cytometer (BDAccuri C6), multichannel pipettor, conjugate: RB-61, RB-66 and RB-70 were all synthesized by Ruibo Biotech, Inc., Guangzhou.

The experimental steps are as follows:

1. diluted GalNac-siRNA

GalNac-siRNA (1uM) was diluted in the following gradient 4-1:

TABLE 4-1

RB-61, RB-66, RB-70 siRNA solutions were prepared according to the above procedure.

2. Cell preparation

Cell concentration 4 x 10 dilution with DMEM medium containing 2% FBS⁵one/mL.

3. Mixed loading and incubation

200uL cells of each sample were mixed with 200uLsiRNA, and 200uL cells of the blank control group were mixed with 200uL of the medium. Add to 3 wells of a U-bottom 96 well plate, 100uL per well. All the siRNA samples with different concentrations are put in an ice box after being added with samples, put on a decoloration shaking table and incubated for 2 hours in a dark place.

4. Detection of

The U-bottom 96-well plate was centrifuged at 50g for 2min at 4 ℃. 80uL of the supernatant was aspirated with a gun, 10ug/mL of Propidium iodide was prepared in PBS containing 2% FBS, and 100uL of PI reagent was added to each well and stained for 15 min. After staining was complete, the U-bottom 96-well plate was centrifuged at 50g for 2min at 4 ℃. After 2 washes with 2% FBS in PBS, 100uL of 2% FBS in PBS was added and resuspended, examined by flow cytometry, and the mean fluorescence intensity of Cy5 fluorescence of the viable cell population was counted to plot a binding saturation curve, as shown in fig. 5.

5. Data analysis

The mean fluorescence intensity of the blank group was subtracted from the mean fluorescence intensity of Cy5 in each experimental group, and the data was fitted using GraphPadprism software to determine the dissociation equilibrium constant Kd, as shown in Table 4-2 below.

TABLE 4-2 affinity Kd values

The data in the table show that the affinity of the siRNA sequences RB-61 and RB-70 linked with the galactose modified monomer L to the mouse primary liver cell is higher than that of RB-66, which indicates that the position linked with the monomer L may have a certain influence on the affinity of the conjugate, and the conjugate is preferably obtained by linking the galactose modified monomer L to the 3' end of the sense chain for modification.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

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Claims

1. An siRNA that inhibits or reduces the expression of a PCSK9 target gene, wherein the siRNA consists of a complementary sense strand and antisense strand;

the base composition sequence of the sense strand is selected from SEQ ID NO.1-SEQ ID NO.2, and the 1 st base from the 5 ' end and the 1 st-2 nd continuous base from the 3 ' end of the sense strand are both subjected to 2' -O-methyl modification;

the base composition sequence of the antisense chain is selected from SEQ ID NO.3-SEQ ID NO. 9.

2. The siRNA for inhibiting or reducing expression of a PCSK9 target gene according to claim 1, wherein in the direction from the 5 'end to the 3' end of the sense strand, bases 1-7 are 2 '-O-methyl modifications and bases 1-7 are 2' -O-methyl modifications;

or, the 1 st and 6 th bases are modified with 2' -O-methyl, the 1 st, 2 th, 5 th and 6 th bases are modified with 2' -O-methyl, and the 4 th and 8 th bases are modified with 2' -fluoro.

3. The siRNA for inhibiting or reducing expression of a PCSK9 target gene according to claim 1, wherein the antisense strand has a 2' -O-methyl modification at the 2 nd base in the 5 ' to 3 ' direction;

or the 3 rd, 4 th, 7 th and 8 th bases are deoxyribonucleotide modifications;

or the 9 th, 11 th, 13 th, 14 th, 15 th, 17 th and 18 th bases are 2' -fluoro modified.

4. siRNA for inhibiting or reducing expression of a PCSK9 target gene according to any one of claims 1 to 3, wherein the base composition sequence of the sense strand is SEQ ID NO.1, and in the direction from the 5 'end to the 3' end of the sense strand, the 1 st, 2 nd, 6 th bases are 2 '-O-methyl modifications, the 4 th, 8 th, 10 th bases are 2' -fluoro modifications, the 1 st, 2 nd, 5 th, 6 th, 8 th bases are 2 '-O-methyl modifications, and the 3 rd, 4 th, 7 th bases are 2' -fluoro modifications;

the base composition sequence of the antisense chain is SEQ ID NO.8, and the 1 st, 4 th, 5 th, 7 th, 8 th, 10 th, 12 th, 13 th, 14 th, 16 th, 17 th, 20 th, 21 th, 22 th and 24 th bases of the antisense chain are 2' -fluoro-modified from the 5 ' end to the 3 ' end.

5. The siRNA for inhibiting or reducing expression of a PCSK9 target gene according to any one of claims 1 to 4, wherein the siRNA for inhibiting or reducing expression of a PCSK9 target gene is modified by a ligand, and the ligand modification is formed by connecting the siRNA with a ligand compound, and the ligand compound has the structure of Ax-linker-R.

6. siRNA for inhibiting or reducing the expression of PCSK9 gene according to claim 5, wherein in the structure of said ligand compound, said Ax is selected from the group consisting of the following structures:

7. siRNA for inhibiting or reducing expression of PCSK9 gene according to any of claims 5-6, wherein said ligand compound has a structure wherein said linker is selected from any of the following structures:

wherein n is an integer from 1 to 10;

or the like, or, alternatively,

wherein n is₁And n₂Each independently selected from an integer of 1 to 10;

or the like, or, alternatively,

wherein n is₁、n₂、n₃Each independently selected from an integer of 1 to 10;

or the like, or, alternatively,

wherein n is an integer from 1 to 10.

8. The siRNA for inhibiting or reducing expression of PCSK9 gene according to any one of claims 5 to 7, wherein in the structure of said ligand compound, said R is selected from R₁、R₂、R₃Any one of, wherein,

R₁is composed of

R₂Is composed of

R₂M in₁And m₂Each independently selected from an integer of 1 to 10;

R₃is composed of

Further, R₁、R₂、R₃Wherein Z is a protecting group for a hydroxyl group, preferably, each Z is independently 4, 4-dimethylOxytrityl or 4-methoxytriphenylchloromethylalkyl.

9. The siRNA for inhibiting or reducing expression of PCSK9 gene according to any of claims 5 to 8, wherein the ligand compound has the structure:

wherein n is an integer from 1 to 10.

10. The siRNA for inhibiting or reducing expression of the PCSK9 gene of claim 9, wherein the ligand compound is modified by conjugation to the siRNA to form any one of the following structures:

wherein Nu is a nucleotide sequence in the siRNA for inhibiting the expression of the PCSK9 gene according to any one of claims 1 to 4.

11. The siRNA for inhibiting or reducing PCSK9 target gene expression according to claim 10, wherein the ligand compound is modified to be ZW1003 or ZW1004 by coordinating with siRNA, wherein n in the ligand compound is 1, Nu is the nucleotide sequence in siRNA for inhibiting or reducing PCSK9 target gene expression according to claim 4.

12. A pharmaceutical composition, wherein the effective ingredient of the pharmaceutical composition comprises the siRNA of any one of claims 1 to 7 for inhibiting or reducing expression of a PCSK9 target gene.

13. The pharmaceutical composition of claim 8, for use in preventing or ameliorating or treating a disease caused by the expression of a PCSK9 target gene.

14. A method for inhibiting or reducing PCSK9 target gene expression, wherein the method is introducing the siRNA of any one of claims 1 to 10 for inhibiting or reducing PCSK9 target gene expression, and/or the pharmaceutical composition of claim 11.

15. The method for inhibiting or reducing the expression of a PCSK9 target gene according to claim 14, wherein the method comprises inhibiting or reducing the expression of a cellular PCSK9 target gene by the steps of:

(1) obtaining a cell to be transfected and siRNA which inhibits or reduces the expression of a PCSK9 target gene according to any one of 1-10;

(2) mixing the siRNA with a cell to be transfected and transfecting the cell;

(3) and (5) culturing the cells to obtain the compound.

16. The method for inhibiting or reducing the expression of a PCSK9 target gene according to claim 15, wherein the siRNA transfection is at a concentration of 4nM to 0.1 μ Μ; more preferably 6nM to 60 nM.